Project Summary/Abstract The broad, long term objectives of this project are to understand the control of human herpesvirus replication and reactivation from latency. Epstein Barr virus (EBV) is a ubiquitous herpesvirus associated with human lymphoid and epithelial malignancies. Lytic replication and expression of lytic gene products during virus reactivation play an important role in pathogenesis. The focus of this application is to investigate the mechanisms of action of SM, an EBV protein that is expressed during lytic replication and is essential for production of infectious virus. SM binds to RNA and enhances expression of several EBV lytic genes including those critical for capsid formation and infectivity. We have shown that these effects are gene-specific and that SM is preferentially required for expression of a subset of EBV genes. A fundamental question is how SM specifically facilitates expression of the mRNAs of these target genes. While SM stabilizes some target RNAs, and has long been thought to only act post-transcriptionally, we provide new evidence that it also facilitates EBV gene transcription. Identifying the targets of these separate mechanisms will allow a molecular dissection of each function. A high throughput screening assay to identify SM inhibitors showed that spironolactone (SPR), a clinically approved mineralocorticoid-blocking agent, has potent anti-SM and antiviral properties. We have found that SPR acts to destabilize a cellular transcription factor, XPB which appears to be uniquely involved in EBV gene transcription. SPR is therefore hypothesized to act through effects on host cell transcription factors that are necessary for SM function. The project has three specific aims. The first is to determine whether SM facilitates transcription initiation/elongation or RNA stabilization to achieve its effects on each of its specific targets. We hypothesize that the combined effect of SM on transcription and mRNA stability of individual genes varies based on specific target gene characteristics. By defining which promoters SM affects, and which RNAs it stabilizes, the mechanisms of transcriptional and post-transcriptional enhancement will be separated and individually investigated. The second aim is to investigate the mechanisms by which spironolactone (SPR) inhibits SM function and to expand its utility as an antiviral agent. Using a synthetic chemistry approach, the mineralocorticoid blocking activity will be ablated from SPR, to demonstrate that it can be separated from antiviral activity. SPR derivatives will also be made to identify its target proteins that will then be identified by proteomic techniques. In the third aim, the unique role of XPB in EBV transcriptional initiation and elongation and its cooperation with SM will be investigated. The combination of these three aims will not only clarify the mechanism of action of SM but will lay the basis for targeting unique interaction points of viral and cellular gene expression for drug development.